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ON THE ANTIBACTERIAL ACTIVITY OF METAL-EXCHANGED
ZEOLITES
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Jelena MilenkovićInnovation Centre of the Faculty of Technology and Metallurgy, University of Belgrade, Serbia
Faculty of Technology and Metallurgy, University of Belgrade, Serbia
Gram-positiveStaphylococcus aureus (Deutsche Sammlung von Microorganismen und Zellkulturen GmbH- DSM 799)
Antibacterial activity
Gram-negativeEscherichia coli
(DSM 498 and isolates from lake water)Acinetobacter baumannii
(clinical isolates belonging to the IC I and II)
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Pathogenic microorganisms
E. coli- indicator of faecal contamination of waters
S. aureus and A. baumannii
Infectious Diseases Society of America-IDSA declared „ESKAPE“
pathogens:
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Countries that have reported an outbreak of carbapenem-resistant Acinetobacter baumannii. Red - outbreaks before 2006, yellow -outbreaks since 2006.
* http://bioquelljp.studiorepublic.com/technology/microbiology/acinetobacter-baumannii/
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Multidrug resistance
Uncontroled use of antibiotic can increase the resistance!
http://www.co-add.org/content/superbug-crisis
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Bacterial cell wall
* Brown L, Wolf J, Prados-Rosales R, Casadevall A, (2015) Nat Rev Microbiol 13:620–630
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Outer membrane
Periplasmicspace
Periplasmicspace
Peptidoglycan
Lipopolysaccaharide
Lipoprotein
Cell membrane
Teihoic acidLipoteihoic acid
Peptidoglycan
Cell membrane
Periplasmicspace
Gram negative bacteria Gram positive bacteria
Disinfection of waters
Physical
Chemical
HeatingUV Membrane proceses(ultrafiltration, reverse osmosis)
ChlorinationO3
Expensive,Formation of cancerogenic by-products
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Expensive
Novel types of disinfectantsNano-particles (TiO2, MgO, Ag, ZnO)Carbon nanotubesChitozanClays and clay mineralsZeolites
Immobilization of E. coli and S. aureus on natural zeolite
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
* Hrenović J, Milenković J, Ivanković T, Rajić N, (2012) J Hazard Mater 201–202:260– 264
Zlatokop mine, Vranjskabanja deposit, Serbia
Metals: ancient antibacterial agents
* Lemire J, Harrison J, Turner R (2013) Nat Rev Microbiol 11:371-384
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
1. Proteine disfunction
- Oxidation of cysteine in Fe-binding site
- Destruction of Fe-S cluster
- Exchange of catalytic metal
- Exchange of structural metal
2. Production of reactive oxygen species (ROS)
3. Impaired membrane function
4. Interference with nutrient uptake
5. Genotoxicity
Mechanism of the antibacterial activity of metal-exchanged zeolites
Two assumptions:Metal-exchanged zeolites generate the production of ROS;Ions leached from the zeolite.
* Matsumura Y, Yoshikata K,Kunisaki S, Tsuchido T, (2003) Appl Environ Microb 69: 4278–4281
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Review of research highlights
Study of antibacterial efficiency of two types of zeolites
Study of antibacterial mechanism
Application of zeolite-based disinfectants
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
CuSO4
ZnSO4 NiSO4
AgNO3
Cu-Z Zn-Z Ni-Z Ag-Z
Ion-exchange procedure
Experimental
Cu-A Zn-A Ni-A Ag-A0.25 mmol M/g zeolite
Natural zeolite (Z) Zeolite A
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Adsorption isotherms and kinetics study of Cu2+
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
tqqkq
t
eet
112
2
+=
Pseudo-second order model:
*Stojaković Đ, Milenković J, Daneu N, Rajić N, (2011) Clay Clay Miner, 59: 277–285
Antibacterial tests
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Treated urban wastewater of the city of Zagreb, Croatia
Water from the Sava lake in Belgrade, Serbia
Synthetic wastewater
Commercial spring water
Phosphate buffer solution (PBS)
Reduction in the numbers of E. coli and S. aureus
Bacteria Medium
Cu-Z Zn-Z Ni-Z
Reduction (%) for different time (1-24 h)
1 24 1 24 1 24
E. coliReal effluent 40.1 93.5 8.09 95.1 1.74 18.5
Synthetic water 19.4 94.9 6.27 93.9 1.38 19.3
S. aureus
Real effluent 55.7 86.8 2.79 82.1 1.18 9.74
Synthetic water 42.8 87.3 3.31 82.3 1.28 10.13
to (E. coli)= 1.1x107 CFU cm-3, to (S. aureus )= 9.9x106 CFU cm-3
* Hrenović J, Milenković J, Ivanković T, Rajić N, (2012) J Hazard Mater 201–202:260– 264
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Metal leaching from zeolite lattices
Sample
Bacteria
E. coli S. aureus
Leachedmetal
(mg dm-3)
Final pH
Leachedmetal
(mg dm-3)Final pH
Cu-Z 0.0 7.14 0.0 6.85
Zn-Z 2.36 7.18 1.58 7.13
Ni-Z 1.79 7.97 4.75 7.90
Maximum allowable concentration in drinking water:Cu - 2 mg dm-3
Zn - 3 mg dm-3
Ni - 0.02 mg dm-3
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
E. coli Medium
Cu-Z Zn-Z Cu-A Zn-A
Reduction (%) for different times (1-24 h)
1 24 1 24 1 24 1 24
Isolate 1
Real effluent 73.6 100 40.3 100 60.7 100 1.37 1.68
Commercial water 84.5 100 82.1 100 83.6 100 0.31 26.4
Isolate 2
Real effluent 60.2 100 10.0 100 51.1 100 3.09 6.68
Commercial water 64.0 100 26.7 100 62.0 100 1.95 14.0
to(Isolate 1) =2.0x107 CFU cm-3, to (Isolate 2)= 2.2x107 CFU cm-3
* Milenkovic J, Hrenovic J, Matijasevic D, Niksic M, Rajic N, (2017) Environ Sci Pollut R, accepted for publication
Reduction in the numbers of E. coli
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
100 100 100
Metal leaching from zeolite lattices
Sample
Isolates of E. coli
Isolate 1 Isolate 2
Leached metal(mg dm-3)
Final pH Leached metal(mg dm-3)
Final pH
Cu-Z 0.30 6.80 0.08 6.78
Zn-Z 0.36 6.85 0.47 6.84
Cu-A 0.06 9.08 0.33 8.82
Zn-A 0.01 9.80 0.01 8.81
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
* Milenkovic J, Hrenovic J, Matijasevic D, Niksic M, Rajic N, (2017) submitted manuscript
TypesSample
Reduction (%) for different time (h) MBC (mg dm-3)
1 24
IC I
Cu-Z 100 100 250
Zn-Z 13.0 28.5 >1000
Ag-Z 100 100 500
IC II
Cu-Z 100 100 125
Zn-Z 10.5 22.0 >1000
Ag-Z 100 100 62.5
0.403
0.0900.103
0.130
Leached metal(mg dm-3)
to (IC I)=8.8x106 CFU cm-3, to (IC II)=1.4x107 CFU cm-3
* Hrenović J, Milenković J, Goić-Barišić I, Rajić N, (2013) Micropor Mesopor Mat 169:148-152
Reduction in the numbers of A. baumannii
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Biofilm
Biofilm of the A. baumannii
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Formation of biofilm
1. Bonding of bacteria cells onto the surface2. Production of extracellular polymer substance 3. Multiplication of bacteria cell inside the biofilm 4. Biofilm “maturation”5. Rupturing of the biofilm and releasing of bacteria.
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
* Stoodley P, Sauer K, Davies D G, Costerton J W, (2002) Annu Rev Microbiol 56:187–209
Formation of biofilm of A. baumannii
Thick layer of extracellular substances with embedded cells of A. baumannii isolates (IC I and II)
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Novel antibiofouling composite
Two series of composites: PVC/Ag-ZPVC/Ag-Z + D-Tyrosine
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Control samplepure PVC
Composite containing max. 15 wt.% of Ag-Z
* Milenković J, Hrenović J, Goić-Barišić I, Tomić M, Đonlagić J, Rajić N, (2014) Biofouling 30:965-973
Reduction of A. baumannii
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Percentage of the Ag released from the composite containing max. amount of Ag-Z over time
SEM image of immobilized A. baumannii on the control surface and the absence of the bacteria
on the Ag-Z1/PVC/D-Tyr
Metal leaching from the composites
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
0 5 10 15 20 250.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Silv
er r
elea
sed,
%Time, h
Rheological studies of composites
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
*Milenković J, Hrenović J, Goić-Barišić I, Tomić M, Đonlagić J, Rajić N, (2014) Biofouling 30:965-973*Milenković J, Hrenović J, Goić-Barišić I, Tomić M, Rajić N, (2015) J Serb Chem Soc(2015) 80: 819–826
Frequency dependences of (a) the storage and loss modulus, and (b) the complex viscosity of the control material and Ag-Z15.
10-1 100 101 102
103
104
G' of Control G' of Ag-Z15
Slope = 2
Sto
rage
modulu
s (G
'),
Pa
Frequency (ω), rad s-1
a)
Slope = 1
102
103
104
G'' of Control G'' of Ag-Z15
Loss
modulu
s (G
''),
Pa
10-1 100 101 102
103
104
Com
ple
x vi
scosi
ty (
η*)
, Pa
s
Frequency (ω), rad s-1
Control Ag-Z15
b)
EDAX elemental mapping of the sample showing C
Typical SEM micrograph of the bacterial sample
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Mechanism of bactericidal activity of Ag+ toward A. baumannii
(left) and Ag distribution (right).
3340 3360 3380 3400
0
2
4
6Ag-citrate
Ag-histidine
Ag-cisteine
Ag-alanine
Ag-imidazole
E (eV)
Ag treated biomass
Norm
aliz
ed a
dso
rption
Ag L3-edge XANES spectra of the Ag-biomass and reference compounds.
XANES measurements
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
XANES measurements
Ag L3-edge XANES spectrum of Ag-biomass: the best linear combination fit obtained by the reference compounds: Ag-alanine, Ag-cysteine, Ag-imidazole and Ag-citrate
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
* Rangus M, Hrenović J, Milenković J, Mazaj M, Sabo T, Rajić N, (2017) J Serb Chem Soc, submitted manuscript
Ag
ON
S
Mechanism of bactericidal activity of Ag+ toward A. baumannii
Genotoxicity Impared membrane function
Ag+
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Conclusions
Metal-exchanged zeolites show good antibacterial activity towards the studied bacterial species.
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017
Natural clinoptilolite can be used alone or in composites for removal of pathogenic bacteria.
Ag-clinoptilolite is perspective antibacterial material for control of multidrug-resistant bacteria.
Acknowledgement
Financial support has been provided by:
Ministry of Education, Science and Technological Development ofthe Republic of Serbia (project no. 172018)
Ministry of Science, Education and Sports of the Republic of Croatia (project no. 1191155-1203); Croatian Science Foundation (project no. IP-2014-09-5656).
7th Slovenian-Serbian-Croatian Symposium on Zeolites, Ljubljana, May 25-27, 2017